Design and development of tissue engineering scafflods using rapid prototyping technology chapter 3

4.1: Water Contact Angle Measurement on Surface Water Droplet Solid Surface θ Figure 3.1: Water contact angle measurement on surface The water contact angle of different polymers PCL,

Chapter Three

Materials and Methods

3.1 Polymer Synthesis

All the polymers used in this study were synthesized at the Centre de Recherche sur les Biopolymères Artificiels (UMR CNRS 5473, Montpellier, France) ∈-Caprolactone was purchased from Acros Organics Ethylene glycol, monohydroxyl PEG (mPEG) and dihydroxyl PEG were supplied by Fluka and used as received DL-lactide (DL-LA) was obtained from Purac (Gorinchem, Holland) and recrystallized from acetone Zinc powder was received from Merck PCL homopolymer, PCL-PEG, PCL-PEG-PCL and PEG-PCL-PLA block copolymers were synthesized by ring-opening polymerization (ROP) of ∈-caprolactone using zinc metal as catalyst as reported elsewhere (Li et al, 1998; Huang et al, 2003)

PCL homopolymer was synthesized by bulk ROP of ∈-caprolactone, using ethylene glycol as initiator and zinc powder as catalyst ∈-Caprolactone and ethylene glycol with a molar ratio of 700/1 and zinc powder (0.05 wt %) were introduced into a round-bottomed flask After degassing at room temperature, the flask was sealed under vacuum and allowed to rotate at 140°C for 11 days

Predetermined amounts of ∈-caprolactone, mPEG or dihydroxyl PEG and zinc powder were introduced into a round-bottomed flask The caprolactone/ethylene oxide or [CL] / [EO] molar ratio was 3/1 Degassing

was performed at 80°C to homogenize the mixture After cooling, the flask was sealed under vacuum and allowed to rotate at 140°C for 7 days

The PEG-PCL-P(DL)LA triblock copolymer was synthesized by stepwise ROP

of ε-caprolactone and DL-lactide In the first step, ε-caprolactone (171 g), mPEG5000 (22 g) and zinc powder (0.05 wt-%) were introduced into a round-bottomed flask Polymerization was carried out under vacuum at 140°C for 9 days The resulting hydroxyl-bearing PEG-PCL was used as macroinitiator in the second step for the ROP of DL-lactide DL-lactide (50 g) was allowed to polymerize in the presence of this PEG-PCL (50 g) and zinc powder (0.05 wt-

%) under vacuum at 140°C for 8 days

All the polymers were recovered by the dissolution/precipitation method with dichloromethane as solvent and ethanol as nonsolvent, followed by filtration and vacuum drying up to constant weight PEG-PCL-PLA block copolymer was further washed with cold acetone (<10°C)

The resulting copolymers were characterized by 1H nuclear magnetic resonance (1H NMR), size exclusion chromatography (SEC), infrared spectroscopy (IR), differential scanning calorimetry (DSC), X-ray diffraction, and thermo gravimetric analysis (TGA)

3.1.5 Measurements

1

H NMR spectra were recorded with a Bruker spectrometer operating at 250MHz, using deuterated chloroform as solvent at room temperature SEC was performed by using a setting composed of a Waters 510 HPLC pump, a Waters 410 differential refractometer and a PL gel 5µm mixed-C 60 cm column, the mobile phase being THF and the flow rate 1 cm3/min Data were expressed with respect to polystyrene standards from Polysciences IR spectra were recorded with a Perkin Elmer 1760 FTIR spectrometer, the film being cast on a NaCl plate from THF solutions DSC measurements were carried out under nitrogen atmosphere from 20°C to 105°C at 5°C/min on a Perkin Elmer Instrument DSC6 thermal analyzer TGA was run on a Perkin Elmer Instrument TGA6 thermal analyzer from 30°C to 450°C at a heating rate of 10°C/min X-ray diffraction spectra were obtained with a Philips apparatus using a Cu Kα source (λ = 0.154 nm)

3.2 Thermal Analyses of Polymers

3.2.1 Differential Scanning Calorimetry (DSC)

Understanding of the melting behavior of the polymer is important to set an appropriate liquefier temperature for scaffold fabrication The thermal characteristics of the polymers before and after processing into scaffolds were determined by differential scanning calorimeter (TA Instruments DSC 2910, New Castle, DE) An indium standard was used to calibrate the instrument The sample weight of around 10 mg was taken All samples were placed in aluminum pans and scanned from 25°C to 95°C at a rate of 5°C/min, using argon as purge gas Five samples for each type of material were analyzed

DSC analysis provided the melting point and degree of crystallinity of the material The degree of crystallinity was calculated according to the following equation:

Xc = (Hm/Hm0) x 100% (3.1) where, Hm is the melting enthalpy of PCL component (only PCL crystallizes in the copolymers) and Hm0 is the melting enthalpy of 100% crystalline PCL (139.5 J/g) (Pitt et al, 1981)

3.2.2 Gel Permeation Chromatography (GPC)

Gel permeation chromatography equipped with a differential refractor (Waters, Model 410, Milford, MA) and an absorbance detector refractor (Waters, Model

2690, Milford, MA) were used to determine the polymer molecular weight distribution The samples were dissolved in tetrahydrofuran (THF) and eluted

in a series of configurations through a Styragel column refractor (Waters, Milford, MA) at a flow rate of 1mL/min Polystyrene standards (Polysciences, Warrington, PA) were used to obtain a calibration curve The GPC experiments were carried out over the polymers before and after fabrication of the scaffold

3.3 Surface Wetability Determination

The surface wetability or hydrophilicity is a specific physical property of any given system that is determined by the contact angle The contact angle is the angle at which a liquid interface meets the solid surface Most often the concept is illustrated with a small liquid droplet resting on a flat horizontal solid

surface The angle between the baseline of the drop and the tangent at the drop boundary is measured as the contact angle as presented in Figure 3.1

4.1: Water Contact Angle Measurement on Surface

Water Droplet

Solid Surface

θ

Figure 3.1: Water contact angle measurement on surface

The water contact angle of different polymers (PCL, PCL-PEG, PCL and PEG-PCL-PLA) was measured on their respective solid flat surfaces

PCL-PEG-by using surface contact angle measuring instrument (VCA Optima XE) Deionized water drop of 5µL was dispensed onto the polymer surface to measure the contact angle Five measurements at five different locations were performed on each polymer sample and the mean values were taken as their respective water contact angles

3.4 Scaffold Processing

3.4.1 Desktop Robot-based RP (DRBRP) Technique

A desktop robot-based rapid prototyping (DRBRP) technique was built in house that consists of a PC-controlled desktop robot (Sony Robot) and a pressure driven dispensing system The robot provides four-axes (Figure 3.2) movement comprising of three simultaneous translational movements along the X-, Y- and Z-axes with an additional rotary motion about the Z-axis The three translational movements had positioning accuracy of up to 0.05mm and

a minimum step resolution of 0.014mm The dispensing system involves a

cylindrical stainless steel chamber with a replaceable extrusion nozzle, melting of scaffold material and extrusion of the melt The stainless steel chamber is insulated by a teflon shell to prevent heat loss because of the developed temperature gradient between heated chamber and the surrounding The heating system comprises of a thermostat-controlled electrical band heater attached to the chamber and a thermocouple (type-K) with an exposed junction inserted near the exit of the chamber, which is used for temperature feedback The extrusion system includes purified compressed air tank and a solenoid valve along with plastic tubing The incoming pressurized air extrudes the semi-molten material through the nozzle that is deposited onto a plastic platform below The heater had the temperature

accuracy of up to 2°C and the feed rate had the deposition speed accuracy of

up to 3mm/min

Material Loading Port

Teflon Insulator

Figure 3.2: Four-axes desktop robot-based rapid prototyping system

3.4.2 Basic Scaffold Modeling Process

The modeling process begins with the creation of a solid shape (e g simple cube) in stl format by CAD software that is imported into slicing software,

RPBOD (in house built) The model is then mathematically cut or sliced into thin horizontal layers in the cli format, followed by the generation of deposition paths according to user-defined parameters within each sliced layer The deposition path data is subsequently downloaded to the robotic machine to build the model using a given material Figure 3.3 schematically summarizes the basic process of preparing the data of a CAD model in RPBOD to fabricate the physical part (scaffold) at the DRBRP machine

Figure 3.3: Outline of the basic DRBRP process Step 1: Import of CAD data

in stl (STereoLithography) format into Sony Robot Step 2: Slicing of the CAD model into horizontal layers and conversion into cli format Step 3: Creation

of desired deposition paths for each layer to upload to the DRBRP machine Step 4: Building of physical model in an additive manner; line-by-line to form a 2D layer and layer-by-layer to form the 3D model

3.4.3 Scaffold Design

Various scaffold architectures can be imprinted by applying various lay-down patterns (0/90°, 0/60/120° and 0/30/60/90/120/150°) as shown in Figure 3.4 using appropriate positioning of the robotic control system The lay-down patterns of 0/90°, 0/60/120° and 0/30/60/90/120/150° are also called 2-angle, 3-angle and 6-angle patterns, respectively

“road” of molten material called, filament with user-defined width and thickness as presented in Figure 3.5 The structural design features that are controllable through slicing flow settings include the road width RW, slice thickness ST, filament gap FG, filament distance and layer gap LG RW is defined as the diameter of the circular cross-section of laid filament, ST is the

vertical distance between the filament centres of adjacent layers, FG is the edge-to-edge horizontal distance between adjacent filaments, FD is the centre-to-centre horizontal distance between adjacent filaments and LG is defined as the edge-to-edge vertical distance between layers of the same filament alignment

Each layer can be filled by rasters, contours or the combination of both The raster fill involves a raster motion of the nozzle within a specified region of a sliced layer There are three ways of raster motions as shown in Figure 3.6; first, one way motion where the polymer-melt is dispended only from one direction of the dispenser movement second, two ways motion where the polymer-melt is dispensed from two opposite directions but still the dispensing

is not connected and third, round-connected way where the polymer is dispensed as connected network from both directions

LG

ΦRW ST

B

A Figure 3.5: Models of lay-down patterns viewed in cross-section A:

0/90°, B: 0/60/120°; Symbols are RW: road width, FG: fill gap,

LG: layer gap, ST: slice thickness

FD

LG

Figure 3.7: One way waster with an additional thin filament

The main advantage of one-way raster motion is that the scaffold edges will

be fully open i.e there will be no blockage of the channels It is an especial

advantage when there needs to build a scaffold with exact dimensions e.g of defect size, without compromising channel connectivity In the two ways motion, the fabrication yield is increased but still there is possibility of producing corrugated edges i.e poor edge finishing On the contrary, the round-connected motion produces high yield along with good edge finishing However, there need to compromise channel connectivity at the edges unless post trimming is carried out Again, the post trimming involves decrease in dimension and material wastage

The contour fill involves several closed loop contour motions of the nozzle inside a specified region or along the periphery of a cross-sectional layer The combination of these two approaches would deposit the molten material within the specified layer by both contour and raster filling as shown in Figure 3.8 The contour fill is necessary for good edge finish and providing adhesion to the raster roads at their ‘U-turns’

Raster

Contour

Figure 3.8: Combination of raster and contour motion

The DRBRP system is also able to pause and continue the material flow during the fabrication of process

The scaffold is built in an additive manner; line-by-line to form a 2D layer and layer-by-layer to form the 3D structure as demonstrated by Figure 3.9 Once a layer is completed the dispenser moves up vertically in the Z-direction by a small displacement equivalent to the specified slice thickness (ST)

escribed in the preceding section In short, the thermoplastic polymers were

r by electrical heating and extruded out

by means of compressed air pressure through a minute nozzle to build 3D scaffolds layer by layer Throughout the study, all the scaffolds were fabricated by employing round-connected raster motion and no additional

0 Layer 0-90 Layers Multiple Layers

contour was drawn other than the U-tern formed due to the round-connected motion Likewise, the scaffolds were fabricated with nozzle size of 500µm internal diameter and FD of 1.5mm unless mentioned otherwise

3.4.4.1 Optimization of Fabrication Process

There are numerous parameters that control the DRBRP system, similar to

DM process as described by Comb et al (1994a), and Agrawal et al (1996)

These parameters can be broadly divided into four cate

in Table 3.1 They are operation specific, machine specific, material specific and geometry specific parameters Most of the parameters are interdependent and have influence on the scaffolds’ morphological and biomechanical properties Therefore, these parameters need to be studied and optimized to produce scaffolds as required The scaffold fabrication was focused on using two polymers namely, PCL and PCL-PEG

Table 3.1: Various Process Parameters in DRBRP Technique

Operation Machine specific Ma

Slice thickness Nozzle tip size Viscosity Fill vector length

Deposition

speed

Extrusion pressure

diameter Liquefier

property

conductivity

In reality all the parameters listed in Table d optim ecise co e fabricati s However, within the scope of this study three major parameters namely, liquefier temperature (TL),

3.1 deserve to be studied anized for pr ntrol of th on proces

extrusion pressure (PE) and deposition speed (SD) were investigated This was primarily to investigate the influence of these parameters on the scaffolds’ morphological and mechanical properties and ultimately to select the best combination of these parameters to develop scaffolds with structural reproducibility and integrity The best selection of process parameters to fabricate 3D scaffold by a solid freeform fabrication technique involves complex interactions among hardware, software and material properties (Comb et al, 1994a) The variation of process parameters produces a range of road widths for a given nozzle diameter In this study, to determine the best-suited values of the process parameters for a given nozzle diameter (e.g 500 µm), it was targeted to achieve raster RW equivalent to nozzle diameter (i.e

500 µm) with minimum fabrication time while structural integrity and reproducibility of the scaffold were maintained This investigation was carried out for two polymers (PCL and PCL-PEG) and one lay-down pattern (0/90) The scaffolds were fabricated employing three values of each parameter for example, liquefier temperature (80, 90 & 100°C), extrusion pressure (3.0, 4.0

& 5.0 bars) and deposition speed (240, 300 & 360 mm/min) Through a series

of trials during which small specimens were fabricated, one parameter was varied iteratively while other two were kept fixed Through this investigation the optimized process parameters were found to be as follows: liquefier temperature of 90°C, extrusion pressure of 4.0 bars and deposition speed of

300 mm/min (see result section 5.5.2 for details) These parameters were set for all further fabrications

Upon optimizing the process parameters, the feasibility of another two ynthetic biopolymers (PCL-PEG-PCL and PEG-PCL-PLA) was investigated

comb like 3D caffolds, the bulk porosity should be above 30% to avoid creation of isolated

s

to process into 3D porous structure with fully interconnected channels This experiment was also to investigate the efficacy of DRBRP system to develop 3D porous scaffolds with a range of polymers, instead of a single one Again, the scaffolds were fabricated with a single lay-down pattern (0/90) employing round-connected raster motion and no additional contour was drawn other than the U-tern formed due to the round-connected motion As all four polymers (PCL, PCL-PEG, PCL-PEG-PCL and PEG-PCL-PLA) have very close melting temperatures (~65°C) for convenience it was decided to apply the same process conditions for them The liquefier temperature, extrusion pressure and deposition speed were set at 90°C, 4.0 bars and 300 mm/min, respectively while the ambient temperature was maintained at 25 ± 2°C Through this investigation the DRBRP system was found to offer the flexibility

to develop 3D porous scaffolds with various synthetic polymers

To achieve completely interconnected pore channels in honey

s

voids (Gibson and Ashby, 1997) Based on this phenomenon, various scaffolds with different values of FD were built up The scaffolds were fabricated with two polymers (PCL and PCL-PEG) and a series of FDs (1.0, 1.25 & 1.5 mm) applying the liquefier temperature, extrusion pressure and

deposition speed of 90°C, 4.0 bars and 300 mm/min, respectively This set of scaffolds was fabricated to investigate the influence of structural design feature, FD on the scaffolds’ morphological and mechanical properties

Lastly, to investigate the variation of morphological characteristics, echanical properties, degradation kinetics and cell culture performances the m

scaffolds were fabricated with three lay-down patterns (0/90°, 0/60/120° and 0/30/60/90/120/150°) using two polymers (PCL and PCL-PEG) The process conditions i.e liquefier temperature, extrusion pressure and deposition speed were set at 90°C, 4.0 bars and 300 mm/min, respectively The microfilaments were laid in coordinate degrees according to the respective lay-down patterns

as generate by the machine toolpath For example, in 0/90° lay-down pattern the filaments were laid as 0° lines and 90° lines Likewise, in 0/60/120° lay-down pattern the filaments were laid as 0° lines, 60° and 120° lines, and in 0/30/60/90/120/150° lay-down pattern the filaments were drawn as 0° lines, 30° lines, 60° lines, 90° lines, 120° lines and 150° lines Overview of the toolpath of the scaffold design process is presented in Figure 3.10 in order

Solid model 0°-line 90°-line

Solid model 0°-line 60°-line 120°-line

Model 0°-line 30°-line 60°-line 90°-line 120°-line 150°-line

Figure 3.10: Overview of scaffold design toolpath; top: 2-angle pattern, middle: 3-angle pattern and bottom: 6-angle pattern

3.4.4.2 Preparation of Test Specimens

The large porous blocks of 50.0 x 50.0 x 5.0 mm were built as initial bulk scaffolds on a flat plastic platform After completion of fabrication the bulk scaffolds were removed from the plastic platform The corrugated edges (Figure 3.11) of the scaffold blocks formed due to the “U-turn” of the round-connected motion of fabrication process were trimmed off to ensure that the entire specimen had uniform edges (Figure 3.11) Then the trimmed scaffolds